Bioactive peptides are small peptides that elicit a biological activity. Since the discovery of secretin in 1902, over 500 of these peptides which average 20 amino acids in size have been identified and characterized. They have been isolated from a variety of systems, exhibit a wide range of actions, and have been utilized as therapeutic agents in the field of medicine and as diagnostic tools in both basic and applied research. Tables 1 and 2 list some of the best known bioactive peptides.
TABLE 1Bioactive peptides utilized in medicineSize InAminoNameIsolated FromAcidsTherapeutic UseAngiotensin IIHuman Plasma8VasoconstrictorBradykininHuman Plasma9VasodilatorCaeruleinFrog Skin10CholereticAgentCalcitoninHuman Parathyroid32CalciumGlandRegulatorCholecystokininPorcine Intestine33CholereticAgentCorticotropinPorcine Pituitary39HormoneGlandEledoisinOctopod Venom11HypotensiveAgentGastrinPorcine Stomach17GastricActivatorGlucagonPorcine Pancreas29AntidiabeticAgentGramicidin DBacillus brevis11AntibacterialBacteriaAgentInsulinCanine PancreasAntidiabeticAgentInsulin A21Insulin B30KallidinHuman Plasma10VasodilatorLuteinizingBovine10HormoneHormone-ReleasingHypothalamusStimulatorFactorMelittinBee Venom26AntirheumaticAgentOxytocinBovine Pituitary9Oxytocic AgentGlandSecretinCanine Intestine27HormoneSermorelinHuman Pancreas29HormoneStimulatorSomatostatinBovine14HormoneHypothalamusInhibitorVasopressinBovine Pituitary9AntidiureticGlandAgent
TABLE 2Bioactive peptides utilized in applied researchSize InAminoNameIsolated FromAcidsBiological ActivityAtrial NatriureticRat Atria28Natriuretic AgentPeptideBombesinFrog Skin14Gastric ActivatorConantokin GSnail Venom17NeurotransmitterConotoxin GISnail Venom13Neuromuscular InhibitorDefensin HNP-1Human30Antimicrobial AgentNeutrophilsDelta Sleep-Rabbit Brain9Neurological AffectorInducing PeptideDermaseptinFrog Skin34Antimicrobial AgentDynorphinPorcine Brain17NeurotransmitterEETI IIEcballium29Protease InhibitorelateriumseedsEndorphinHuman Brain30NeurotransmitterEnkephalinHuman Brain5NeurotransmitterHistatin 5Human Saliva24Antibacterial AgentMastoparanVespid Wasps14Mast Cell DegranulatorMagainin IFrog Skin23Antimicrobial AgentMelanocytePorcine13Hormone StimulatorStimulatingPituitaryHormoneGlandMotilinCanine22Gastric ActivatorIntestineNeurotensinBovine Brain13NeurotransmitterPhysalaeminFrog Skin11Hypotensive AgentSubstance PHorse11VasodilatorIntestineVasoactivePorcine28HormoneIntestinalIntestinePeptideWhere the mode of action of these peptides has been determined, it has been found to be due to the interaction of the bioactive peptide with a specific protein target. In most of the cases, the bioactive peptide acts by binding to and inactivating its protein target with extremely high specificities. Binding constants of these peptides for their protein targets typically have been determined to be in the nanomolar (nM, 10−9 M) range with binding constants as high as 10−12 M (picomolar range) having been reported. Table 3 shows target proteins inactivated by several different bioactive peptides as well as the binding constants associated with binding thereto.
TABLE 3Binding constants of bioactive peptidesSize inBioactiveAminoPeptideAcidsInhibited ProteinBinding Constantα-Conotoxin GIA15Nicotinic1.0 × 10−9 MAcetylcholineReceptorEETI II29Trypsin1.0 × 10−12 MH2(7-15)8HSV3.6 × 10−5 MRibonucleotideReductaseHistatin 524Bacteroides5.5 × 10−8 MgingivalisProteaseMelittin26Calmodulin3.0 × 10−9 MMyotoxin (29-42)14ATPase1.9 × 10−5 MNeurotensin13Ni Regulatory5.6 × 10−11 MProteinPituitary Adenylate38Calmodulin1.5 × 10−8 MCyclase ActivatingPolypeptidePKI (5-24)20cAMP-Dependent2.3 × 10−9 MProtein KinaseSCP (153-180)27Calpain3.0 × 10−8 MSecretin27HSR G Protein3.2 × 10−9 MVasoactive28GPRN1 G Protein2.5 × 10−9 MIntestinal Peptide
Recently, there has been an increasing interest in employing synthetically derived bioactive peptides as novel pharmaceutical agents due to the impressive ability of the naturally occurring peptides to bind to and inhibit specific protein targets. Synthetically derived peptides could be useful in the development of new antibacterial, antiviral, and anticancer agents. Examples of synthetically derived antibacterial or antiviral peptide agents would be those capable of binding to and preventing bacterial or viral surface proteins from interacting with their host cell receptors, or preventing the action of specific toxin or protease proteins. Examples of anticancer agents would include synthetically derived peptides that could bind to and prevent the action of specific oncogenic proteins.
To date, novel bioactive peptides have been engineered through the use of two different in vitro approaches. The first approach produces candidate peptides by chemically synthesizing a randomized library of 6–10 amino acid peptides (J. Eichler et al., Med. Res. Rev. 15:481–496 (1995); K. Lam, Anticancer Drug Des. 12:145–167 (1996); M. Lebl et al., Methods Enzymol. 289:336–392 (1997)). In the second approach, candidate peptides are synthesized by cloning a randomized oligonucleotide library into a Ff filamentous phage gene, which allows peptides that are much larger in size to be expressed on the surface of the bacteriophage (H. Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401–424 (1997); G. Smith et al., et al. Meth. Enz. 217:228–257 (1993)). To date, randomized peptide libraries up to 38 amino acids in length have been made, and longer peptides are likely achievable using this system. The peptide libraries that are produced using either of these strategies are then typically mixed with a preselected matrix-bound protein target. Peptides that bind are eluted, and their sequences are determined. From this information new peptides are synthesized and their inhibitory properties are determined. This is a tedious process that only screens for one biological activity at a time.
Although these in vitro approaches show promise, the use of synthetically derived peptides has not yet become a mainstay in the pharmaceutical industry. The primary obstacle remaining is that of peptide instability within the biological system of interest as evidenced by the unwanted degradation of potential peptide drugs by proteases and/or peptidases in the host cells. There are three major classes of peptidases which can degrade larger peptides: amino and carboxy exopeptidases which act at either the amino or the carboxy terminal end of the peptide, respectively, and endopeptidases which act on an internal portion of the peptide. Aminopeptidases, carboxypeptidases, and endopeptidases have been identified in both prokaryotic and eukaryotic cells. Many of those that have been extensively characterized were found to function similarly in both cell types. Interestingly, in both prokaryotic and eukaryotic systems, many more aminopeptidases than carboxypeptidases have been identified to date.
Approaches used to address the problem of peptide degradation have included the use of D-amino acids or modified amino acids as opposed to the naturally occurring L-amino acids (e.g., J. Eichler et al., Med Res Rev. 15:481–496 (1995); L. Sanders, Eur. J. Drug Metabol. Pharmacokinetics 15: 95–102 (1990)), the use of cyclized peptides (e.g., R. Egleton, et al., Peptides 18:1431–1439 (1997)), and the development of enhanced delivery systems that prevent degradation of a peptide before it reaches its target in a patient (e.g., L. Wearley, Crit. Rev. Ther. Drug Carrier Syst. 8: 331–394 (1991); L. Sanders, Eur. J. Drug Metabol. Pharmacokinetics 15: 95–102 (1990)). Although these approaches for stabilizing peptides and thereby preventing their unwanted degradation in the biosystem of choice (e.g., a patient) are promising, there remains no way to routinely and reliably stabilize peptide drugs and drug candidates. Moreover, many of the existing stabilization and delivery methods cannot be directly utilized in the screening and development of novel useful bioactive peptides. A biological approach that would serve as both a method of stabilizing peptides and a method for identifying novel bioactive peptides would represent a much needed advance in the field of peptide drug development.